EGFR-targeted granzyme B expressed in NK cells enhances natural cytotoxicity and mediates specific killing of tumor cells

Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells

Selective Induction of Cancer Cell Death by Targeted Granzyme B

The potential utility of immunotoxins for cancer therapy has convincingly been demonstrated in clinical studies. Nevertheless, the high immunogenicity of their bacterial toxin domain represents a critical limitation, and has prompted the evaluation of cell-death inducing proteins of human origin as a basis for less immunogenic immunotoxin-like molecules. In this review, we focus on the current status and future prospects of targeted fusion proteins for cancer therapy that employ granzyme B (GrB) from cytotoxic lymphocytes as a cytotoxic moiety. Naturally, this serine protease plays a critical role in the immune defense by inducing apoptotic target cell death upon cleavage of intracellular substrates. Advances in understanding of the structure and function of GrB enabled the generation of chimeric fusion proteins that carry a heterologous cell binding domain for recognition of tumor-associated cell surface antigens. These hybrid molecules display high selectivity for cancer cells, with cell killing activities similar to that of corresponding recombinant toxins. Recent findings have helped to understand and circumvent intrinsic cell binding of GrB and susceptibility of the enzyme to inhibition by serpins. This now allows the rational design of optimized GrB derivatives that avoid sequestration by binding to non-target tissues, limit off-target effects, and overcome resistance mechanisms in tumor cells

Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

Background: The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings: We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal a-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furinlike proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions: Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable t

Directed differentiation of mobilized hematopoietic stem and progenitor cells into functional NK cells with enhanced antitumor activity

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy

Release of GrB-T protein upon degranulation of NK cells.

<p>(A) Degranulation of NKL, NKL/GrB-T and NKL/GrB_S183A-T cells was induced by treatment with PMA and ionomycin for 5 h at 37°C, and culture supernatants were harvested. To confirm activation of cells, CD107a expression was analyzed by flow cytometry (open areas). Unstimulated cells served as controls (shaded areas). (B) To determine enzymatic activity of GrB and GrB-T proteins, GrB-specific peptide substrate Ac-IETD-pNA was incubated with 50 to 200 µg/mL of total proteins from supernatants of activated NKL (filled circles), NKL/GrB-T (filled squares) or NKL/GrB_S183A-T cells (open squares). Substrate cleavage was determined by measuring the absorbance at 405 nm. Mean values ± SEM are shown; n = 4. *, <i>P</i><0.05. (C) Binding of GrB-T (bold line) and mutant GrB_S183A-T protein (dotted line) released by activated NKL/GrB-T and NKL/GrB_S183A-T cells to EGFR-positive MDA-MB468 and EGFR-negative MDA-MB453 breast carcinoma cells was determined by flow cytometry with GrB-specific antibody. Cells treated with medium (shaded areas) or proteins released by activated parental NKL cells (regular line) served as controls.</p

Cell binding and cytotoxicity of GrB-T fusion protein towards A431 cells.

<p>(A) Binding of GrB-T (bold line) and mutant GrB_S183A-T protein (dotted line) released by activated NKL/GrB-T and NKL/GrB_S183A-T cells to EGFR-positive A431 squamous cell carcinoma cells was determined by flow cytometry with GrB-specific antibody. Cells treated with medium (shaded areas) or proteins released by activated parental NKL cells (regular line) served as controls. (B) To determine cytotoxicity, A431 cells were treated with 100 µg/mL of total proteins from supernatants of activated NKL, NKL/GrB-T, or NKL/GrB_S183A-T cells in the presence of 100 µM chloroquine as indicated. Controls cells were treated with medium containing PMA, ionomycin and chloroquine. After 24 h, the relative number of viable cells was determined in WST-1 assays. Mean values ± SEM are shown; n = 3. **, <i>P</i><0.01; *, <i>P</i><0.05; ns, <i>P</i>>0.05.</p

Natural cytotoxicity of NKL/GrB-T and NKL/GrB_S183A-T cells.

<p>(A) Total levels of GrB and perforin expressed by parental NKL (dark gray areas), NKL/GrB-T (bold lines) and NKL/GrB_S183A-T cells (dotted lines) was analyzed by intracellular staining with GrB-specific antibody (left) or perforin-specific antibody (right) and flow cytometry. NKL cells incubated with isotype-matched antibodies served as controls (light gray areas). (B) Cytotoxicity of NKL/GrB-T (filled squares) and NKL/GrB_S183A-T cells (open squares) towards C1R-neo and Jurkat cells was determined in FACS-based cytotoxicity assays at different effector to target ratios (E/T). Parental NKL cells (filled circles) were included for comparison. Dependence of target cell killing on the release of granular proteins was confirmed by incubating C1R-neo target cells with NKL effector cells in the presence of 2 mM of the Ca²⁺ chelator EGTA. Representative data of one of three independent experiments are shown. Absence of EGFR expression on the surface of C1R-neo and Jurkat cells was confirmed by flow cytometry with EGFR-specific antibody (open areas). Cells treated only with secondary antibody served as controls (shaded areas).</p